During glycolysis, cells convert simple sugars, such as glucose, into pyruvic acid, with a net production of ATP and NADH. In the absence of a functioning electron transport system for oxidative phosphorylation, at least 95% of the pyruvic acid is consumed in short pathways which regenerate NAD+, an obligate requirement for continued glycolysis and ATP production. The waste products of these NAD+ regeneration systems are commonly referred to as fermentation products.
In most animals and plants as well as bacteria, yeast, and fungi, glucose is degraded initially by an anaerobic pathway prior to either oxidative or fermentative metabolism. The most common such pathway, termed glycolysis, refers to the series of enzymatic steps whereby the six-carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound, pyruvate. During this process, two molecules of NAD+ are reduced to form NADH. The net reaction in this transformation of glucose into pyruvate is:glucose+2 Pi+2 ADP+2 NAD+→2 pyruvate+2 ATP+2 NADH+2 H+
For glycolysis to continue, the NAD+ consumed by glycolysis must be regenerated by the oxidation of NADH. During oxidative metabolism, NADH typically is oxidized by donating hydrogen equivalents via a series of steps to oxygen, thereby forming water. Most organisms contain additional anaerobic pathways, however, which allow glycolysis to continue in the absence of compounds like oxygen. Such anaerobic processes are termed fermentation, and homolactic fermentation is perhaps one of the most common of these pathways occurring in many bacteria and in animals. In homolactic fermentation, glucose ultimately is converted to two molecules of the three-carbon acid, lactic acid.
Ethanologenic organisms like Zymomonas mobilis and Saccharomyces cerevisiae are capable of a second (alcoholic) type of fermentation whereby glucose is metabolized to two molecules of ethanol and two molecules of CO2. Alcoholic fermentation differs from lactic acid fermentation in the steps used for the regeneration of NAD+. Two different enzymatic steps are required for alcoholic fermentation. Pyruvate decarboxylase cleaves pyruvate into acetaldehyde and carbon dioxide. Alcohol dehydrogenase serves to regenerate NAD+ by transferring hydrogen equivalents from NADH to acetaldehyde, thereby producing ethanol. The reactions for the regeneration of NAD+ by alcoholic fermentation are:2 Pyruvate→2 Acetaldehyde+2 CO2 2 Acetaldehyde+2 NADH→2 Ethanol+2 NAD+
The net reaction for alcoholic fermentation is:2 Pyruvate+2 NADH→2 Ethanol+2 CO2+2 NAD+
Pentose sugars, which can also be converted to ethanol, are abundant in nature as a major component of lignocellulosic biomass. One such pentose sugar is xylose, which is second only to glucose in natural abundance. Thus, as with hexose sugars, pentose sugars such as xylose can be converted into pyruvate by modified glycolytic pathways. The pyruvate can then be redirected to ethanol. The net reaction for a pentose sugar is typically: three pentose sugars yield five ethanol and five carbon dioxide molecules. Because of the abundance of pentose sugars, the fermentation of xylose and other hemicellulose constituents is an attractive option for the development of an economically viable process to produce ethanol from biomass. However, no naturally occurring microorganisms have been found which rapidly and efficiently ferment pentoses to high levels of ethanol. Yeasts such as Pachysolen tannophilus, Candida shehatae, and Pichia stipitis have been investigated as candidates for xylose fermentation. Efficient fermentation by these pentose-fermenting yeasts has proven difficult due to a requirement for oxygen during ethanol production, acetate toxicity, and the production of xylitol as a by-product. Other approaches to xylose fermentation include the conversion of xylose to xylulose using xylose isomerase prior to fermentation by Saccharomyces cerevisiae (Gong et al., 1981) and the development of genetically engineered strains of S. cerevisiae which express xylose isomerase (Sarthy et al., 1987). The thermophilic bacterium, Clostridium thermosaccharolyticum, represent an alternative and promising approach to xylose fermentation (Mistry and Cooney, 1989 [p. 1295]; Mistry and Cooney, 1989 [p. 1305]). High volumetric productivities have been achieved in continuous culture although final ethanol concentrations remained low.
Microorganisms are particularly diverse in the array of fermentations products which are produced by different genera (Krieg, N. R., and J. G. Holt, eds. [1984] Bergey's manual of systematic bacteriology, The Williams & Wilkins Co., Baltimore). These products include organic acids, such as lactic, acetic, succinic, and butyric, as well as neutral products, such as ethanol, butanol, acetone, and butanediol. Indeed, the diversity of fermentation products from bacteria has led to their use as a primary determinant in taxonomy (Krieg and Holt [1984], supra).
End products of fermentation share several fundamental features. They are relatively nontoxic under the conditions in which they are initially produced but become more toxic upon accumulation. The microbial production of these fermentation products forms the basis for our oldest and most economically successful applications of biotechnology and includes dairy products, meats, beverages, and fuels. In recent years, many advances have been made in the field of biotechnology as a result of new technologies which enable researchers to selectively alter the genetic makeup of some microorganisms. The invention described here relates to the use of recombinant DNA technology to elicit the production of specific useful products by a modified host.
The DNA used to modify the host of the subject invention can be obtained from Zymomonas mobilis. Z. mobilis is a microorganism which is commonly found in plant saps and in honey, and which has unusual metabolic characteristics. Z. mobilis has long served as a natural inocula for the fermentation of the Agave sap to produce pulque (an alcohol-containing Mexican beverage) and as inocula for palm wines. This organism is also used for fuel ethanol production and has been reported capable of ethanol production rates which are substantially higher than that of yeasts.
Although Z. mobilis is nutritionally simple and capable of synthesizing amino acids, nucleotides and vitamins, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose. Z. mobilis is incapable of growth even in rich medium such as nutrient broth without a fermentable sugar.
Like the yeast Saccharomyces cerevisiae, Z. mobilis produces ethanol and carbon dioxide as principal fermentation products. Z. mobilis produces ethanol by a short pathway which requires only two enzymatic activities: pyruvate decarboxylase and alcohol dehydrogenase. Pyruvate decarboxylase is the key enzyme in this pathway which diverts the flow of pyruvate to ethanol. Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Two alcohol dehydrogenase isozymes are present in this organism and catalyze the reduction of acetaldehyde to ethanol during fermentation, accompanied by the oxidation of NADH to NAD+. Although bacterial alcohol dehydrogenases are common in many organisms, few bacteria have pyruvate decarboxylase. Attempts to modify Z. mobilis to enhance its commercial utility as an ethanol producer have met with very limited success.
Most fuel ethanol is currently produced from hexose sugars derived from corn starch or cane syrup utilizing either S. cerevisiae or Z. mobilis. However, these are relatively expensive sources of biomass sugars and have competing value as foods. Starches and sugars represent only a fraction of the total carbohydrates in plants. The majority of the world's cheap, renewable source of biomass is not found as monosaccharides but rather in the form of lignocellulose, which is primarily a mixture of cellulose, hemicellulose, and lignin. The dominant forms of plant carbohydrate in stems, leaves, hulls, husks, cobs, etc. are the structural wall polymers, cellulose and hemicellulose. Hydrolysis of these polymers releases a mixture of neutral sugars which include glucose, xylose, mannose, galactose, and arabinose. Cellulose is a homopolymer of glucose, while hemicellulose is a more complex heteropolymer comprised not only of xylose, which is its primary constituent, but also of significant amounts of arabinose, mannose, glucose, and galactose. No single organism has been found in nature which can rapidly and efficiently metabolize these sources of biomass into ethanol or any other single product of value.
It has been estimated that microbial conversion of the sugar residues present in waste paper and yard trash from U.S. landfills could provide over ten billion gallons of ethanol. While microorganisms such as those discussed above can ferment efficiently the monomeric sugars which make up the cellulosic and hemicellulosic polymers present in lignocellulose, the development of improved methods for the saccharification of lignocellulose remains a major research goal.
Current methods of saccharifying lignocellulose include acidic and enzymatic hydrolyses. Acid hydrolysis usually requires heat and presents several drawbacks, including the use of energy, the production of acidic waste, and the formation of toxic compounds which can hinder subsequent microbial fermentations. Enzymatic hydrolysis thus presents a desirable alternative. For example, enzymes can be added directly to the medium containing the lignocellulosic material while microorganisms are growing therein.
Genetic-engineering approaches for the addition of saccharifying traits to microorganisms for the production of ethanol or lactic acid have been directed at the secretion of high enzyme levels into the medium. That is, the art has concerned itself with modifying microorganisms already possessing the requisite proteins for transporting cellularly-produced enzymes into the fermentation medium, where those enzymes can then act on the polysaccharide substrate to yield mono- and oligosaccharides. This approach has been taken because the art has perceived difficulty in successfully modifying organisms lacking the requisite ability to transport such proteins.
The genes encoding alcohol dehydrogenase II and pyruvate decarboxylase in Z. mobilis have been separately cloned, characterized, and expressed in E. coli. See Bräu & Sahm (1986a) Arch. Microbiol. 144:296-301, (1986b) Arch. Microbiol. 146:105-110; Conway et al. (1987a) J. Bacteriol. 169:2591-2597; Neale et al. (1987) Nucleic Acids Res. 15:1752-1761; Ingram and Conway [1988] Appl. Environ. Microbiol. 54:397-404; Ingram et al. (1987) Appl. Environ. Microbiol. 53:2420-2425.
Bräu and Sahm (1986a), supra, first demonstrated that ethanol production could be increased in recombinant E. coli by the over-expression of Z. mobilis pyruvate decarboxylase although very low ethanol concentrations were produced. Subsequent studies extended this work by using two other enteric bacteria, Erwinia chrysanthemi and Klebsiella planticola, and thereby achieved higher levels of ethanol from hexoses, pentoses, and sugar mixtures. See Tolan and Finn (1987) Appl. Environ. Microbiol. 53:2033-2038, 2039-2044. The genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis have been expressed at high levels in Gram-negative bacteria, effectively redirecting fermentative metabolism to produce ethanol as the primary product (Beall et al., 1993; Ingram and Conway, 1988; Wood and Ingram, 1992).
Prior to our work, there has been no report of the transformation of Gram-positive bacteria to produce ethanol. The presence of multiple proteinases with overlapping specificities in Bacillus has been well established (Koidë et al., 1986; O'Hara and Hageman, 1990) and may limit high level expression.